Optimizing diagnostics for galactofuranose containing antigens

ABSTRACT

Disclosed herein are methods of detecting microbial infection in mammalian subjects comprising treatment of a sample and detection of galactofuranose (galF)-containing antigenic components utilizing monoclonal antibodies. The methods disclosed provide for pretreatment of biological samples, such as urine samples, to maximize detection of galF antigens and improvement of sensitivity of galF antigen detection assays. The methods include minimizing intelectin-1 binding to galF antigens and improvement of monoclonal antibody binding. The detection methods are useful for identifying the presence of microbial antigens related to bacterial, fungal, and parasitic pathogens, including Streptococcus pneumoniae, Aspergillus species, Fusarium species, Coccidioides species, Cryptococcus species, Histoplasma species, and Leishmania species.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 15/882,278, filed Jan. 29, 2018, which is acontinuation-in-part application of U.S. patent application Ser. No.14,546,830, filed on Nov. 18, 2014 which is a Continuation of U.S.patent application Ser. No. 13/511,264, filed on Sep. 25, 2012, which isa § 371 application of International Application No. PCT/US2010/057819,filed Nov. 23, 2010, which claims priority to U.S. Provisional PatentApplication No. 61/263,498, filed Nov. 23, 2009, each of which arehereby incorporated by reference for all purposes as if fully set forthherein.

This invention was made with government support under grant numbersAI054736, AI053623, AI065745, awarded by the National Institutes ofHealth. The government has certain rights in the invention.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 27, 2020, isnamed 048317-551C03US_SL.txt and is 6,931 bytes in size.

BACKGROUND OF THE INVENTION

Galactose is common in mammals, but only found in the 6-member ringhexopyranosyl form, called galactopyranose (galP). Galactofuranose(galF), the 5-member ring form of galactose, is found in otherorganisms, including bacteria, fungi, protozoa, lichens, green algae,starfish and sponges. Equilibrium strongly favors the galP form unlessthe organism contains specific enzymes to catalyze maintenance of galF.In these organisms, galF is an important residue on glycoconjugateantigens, and can be found linked to secreted and cellularpolysaccharides, glycoproteins and glycosphingolipids. In certainorganisms where the galF form is detected, introduction of galF is viainhalation and pulmonary introduction.

The present inventors previously identified a class of antibodies thatwere generated against conidia of an important fungal pathogen, calledAspergillus fumigatus. These antibodies were found to identifygalF-containing antigens that were quickly excreted in urine afterinfection in mammals. The antibodies and the technology enable their useas a urine diagnostic assay.

What is needed however, are methods for improving detection andoptimizing the sensitivity of the antibodies, namely improved detectionof galF-containing antigens in biological samples such as urine. What isalso needed are methods that improve sensitivity and performance of suchdetection assays with minimal sample processing.

SUMMARY OF THE INVENTION

In accordance with one or more embodiments, the present inventionprovides methods for improved galF antigen detection in human bodyfluids by disabling or otherwise removing a competitive inhibitor, thehuman lectin, intelectin-1 from the assay process previously disclosedin U.S. patent application Ser. No. 13/511,264, and incorporated byreference herein in its entirety. Although intelectin is known to besecreted by epithelial cells lining the GI tract and respiratory tract,and for recognizing galF antigens, intelectin was not known until now tobe present in urine. The present inventors surprisingly determined thatintelectin is also present in urine, and serves to compete withgalF-directed antibodies when they are used as part of diagnosingmicrobial infections in a mammalian subject. Though not wishing to bebound by the following theory, it is thought that this inhibition orinterference is dependent on calcium, which can be removed by chelationas a mechanism to optimize antigen identification.

In some embodiments, the microbial infection is caused by an organismselected from the group consisting of Streptococcus pneumoniae,Aspergillus species, Fusarium species, Coccidioides species,Cryptococcus species, and Histoplasma species.

In some embodiments, the microbial infection is caused by Prokaryoticand Eukaryotic pathogens that produce galF-containing antigens,including but not limited to, Streptococcus species, Pseudomonasspecies, Nocardia species, Actinomyces species. Zygomycetes andparasites, including but not limited to Leishmania species, andTrypanosoma species.

As such, in accordance with one or more embodiments, the presentinvention provides methods for optimization of galF-antigenidentification in fluids that contain intelectin, including urine,respiratory fluids, gastrointestinal fluids, and blood. The presentinventors found that this is important for optimizing those methods thatspecifically focus on fungal antigens. The utility is broadly applicableto diagnostics that target galF containing antigens in many differentdiagnostic systems, given the ubiquity of galF in microbial antigens.

In accordance with an embodiment, the present invention provides amethod for diagnosing a microbial infection in a biological sample froma mammalian subject suspected of having, having, or susceptible tohaving a microbial infection by detecting the presence of at least onemicrobial molecule comprising a galactofuranose (galF) residue (forexample, glycoprotein, polysaccharide or sphingolipid) in a biologicalsample of the mammalian subject, the method comprising: (a) treating thebiological sample to decrease human intelectin (hIntL) binding of galFresidues present in the sample; (b) allowing the treated sample of (a)to come in contact with at least one antibody specific for at least onemolecule comprising a galF residue in an effective amount to produce adetectable amount of antibody-galF complex; and (c) detecting thepresence of at least one antibody-galF complex, wherein the detection ofthe presence of at least one antibody-galF complex is diagnostic of amicrobial infection in a mammalian subject.

In accordance with another embodiment, the present invention provides amethod for diagnosing a microbial infection in a biological sample froma mammalian subject suspected of having, having, or susceptible tohaving a microbial infection by detecting the presence of at least oneantigen comprising a galF residue in a biological sample of themammalian subject, the method comprising: (a) treating the biologicalsample comprising contacting the sample with a substrate such as aligand which binds directly to intelectin, or calcium or divalentcations with high affinity, thereby inhibiting or interfering with humanintelectin (hIntL) binding of microbial molecule containing galFresidues present in the sample; (b) contacting the treated sample of (a)with at least one antibody specific for at least one molecule comprisinga galF residue in an effective amount to produce a detectable amount ofantibody-galF complex; and (c) detecting the presence of at least oneantibody-galF complex, wherein the detection of the presence of at leastone antibody-galF complex is diagnostic of a microbial infection in amammalian subject.

In accordance with some embodiments, the method for treating the samplein step (a) comprises contacting the sample with a substrate which bindsCa²⁺ ions with high affinity.

In accordance with some embodiments, the method for treating the samplein step (a) comprises contacting the sample with a compound whichchelates Ca²⁺ ions with high affinity.

In accordance with some embodiments, the method for treating the samplein step (a) comprises contacting the sample with EDTA and/or EGTA.

In accordance with some embodiments, the method for treating the samplein step (a) comprises contacting the sample with a substrate which bindshIntL with high affinity.

In accordance with some embodiments, the method for treating the samplein step (a) comprises contacting the sample with an antibody specificfor hIntL.

In accordance with some embodiments, the method for treating the samplein step (a) comprises contacting the sample with one or more compoundswhich are bound by hIntL with high affinity, thereby preventinggalF-binding to hIntL.

In accordance with some embodiments, the method for treating the samplein step (a) comprises contacting the sample with one or more compoundswhich are bound by hInL with high affinity selected from the groupconsisting of glycerol, 3-Keto-2-deoxyoctonic acid;D-glycerol-1-phosphate, D-mannoheptose, sepharose, sepharose-containingparticles (i.e. latex, polystyrene or glass beads, microspheres orgels).

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 illustrates the inventors' prior discovery of galF antigens inthe urine of patients with Aspergillus infection and production ofmonoclonal antibodies specific for galF antigens, with sensitivity todetect excreted antigens in urine samples.

FIG. 2 shows intelectin and what is known about lectin molecules.

FIG. 3 shows the inventors' hypothesis that hIntL was secreted into theurine and was competitively binding to galF antigens in the presence ofthe inventors' monoclonal antibodies specific for galF, and thatdesalting removes Ca²⁺ needed for hIntL to bind galF.

FIGS. 4A-4B depict (A) recombinant hIntL-1 binds to Aspergillus ethanolprecipitate (EP) in an ELISA, and (B) binds to whole cells (conidia andhyphae).

FIG. 5 shows the presence of hIntL-1 in urine from aspergillosis patientand healthy control. Bands with rabbit anti-IntL antibody seen only inlane from concentrated urine. VU7=aspergillosis patient; Pool4/P4=healthy control urine; “c” denotes concentrated urine via Amicon(10 kDa MWCO); “ds” denotes treatment with desalting column. The gelswere exposed for 5 s to SuperSignal West Pico Chemiluminescent reagent(ThermoFisher Inc.).

FIG. 6 shows that urine from a patient with invasive Aspergillusinfection (aspergillosis) contains a different conformation of hIntLthan healthy control urine. Radiograms from Western blots show that theaspergillosis patient urine contains small molecular weight bands whichmigrate at sizes consistent with subunits of the hIntL-1 heterotrimerprotein. Healthy patient urine contains a single 120 kDa band of hIntL,consistent with the intact heterotrimer.

FIG. 7 shows that galF specific antibody mAB476 binds to the band thatco-migrates with that recognized by anti-hIntL and shows that when theaspergillosis patient urine is desalted prior to contacting the urinewith the antibody, the amount of antibody binding is increased.Desalting also appears to affect the hIntL heterotrimer protein as moreof it is found in small molecular weight bands which are the subunits ofhIntL.

FIG. 8 depicts small to medium molecular weight proteins on SDS-PAGE gelprobed with mAB476 at a 1:1000 titer and detected with goat-anti-mouseIgM-HRP at a titer of 1:10,000. The mAB476 antibody binds to these smallmolecular weight moieties in aspergillosis patient urine, but not inhealthy patient urine; these are the galF-containing antigens that arereleased by the microbe.

FIG. 9 shows localization of mAB476 antibody-reactive bands relative tothe anti-hIntL antibody reactive bands.

FIG. 10 depicts a reducing SDS-PAGE gels of concentrated urine fromhealthy (pool 4) and patient (VU7), both normal and desalted, were runand stained with Sypro-Ruby fluorescent protein stain. The small tomid-range molecular weight bands were cut out of the gels and preparedfor mass spectrometry analysis.

FIG. 11 shows graphs depicting immunoprecipation assays using mAB476 inurine samples of urine samples, both normal and desalted. To enrichsample detection of moieties specifically binding to mAB476, urines wereincubated with superparamagnetic dynabeads coated with mAB476 and amouse control antibody. mAB476 reactive material was eluted with 50 mMglycine (pH 2.5-3.0). No mAB476 binding occurred in normal urine.Reactivity to mAB476 eluates was confirmed by sandwich ELISA. Eluateswere then run on SDS-PAGE gels to check for band patterns, andin-solution mass spectrometry analysis was performed. Analysis detectedboth the microbial antigen CelA/Aspf7 (a galF-containing O-glycan), andthe human hIntL protein (FIG. 12).

FIGS. 13 and 14 show that treatment of the patient urine with calciumchelators or other compounds which bind calcium or compete with galF forhIntL-1 binding with high affinity, when added prior to contacting thesample with galF antibodies, significantly improves detectionsensitivity.

DETAILED DESCRIPTION OF THE INVENTION

The following detailed description is exemplary and explanatory and isintended to provide further explanation of the present disclosuredescribed herein. Other advantages, and novel features will be readilyapparent to one of ordinary skill in the art from the following detaileddescription of the present disclosure.

Disclosed herein are methods of detecting microbial infection inmammalian subjects comprising treatment of a sample and detection ofpolysaccharide antigenic components utilizing monoclonal antibodies. Themethods disclosed provide for pretreatment of biological samples, suchas urine samples, to maximize detection of galF antigens and improvementof sensitivity of galF antigen detection assays. The methods includeminimizing hIntL binding to galF antigens and improvement of monoclonalantibody binding. The detection methods are useful for identifying thepresence of microbial antigens related to Streptococcus pneumoniae,Aspergillus species, Fusarium species, Coccidioides species,Cryptococcus species, and Histoplasma species. In some embodiments, themicrobial infection is caused by Prokaryotic and Eukaryotic pathogensthat produce galF-containing antigens, including but not limited to,Streptococcus species, Pseudomonas species, Nocardia species,Actinomyces species. Zygomycetes and parasites, including but notlimited to Leishmania species, and Trypanosoma species.

Intelectin was previously called ‘omentin’ as it is secreted fromadipose cells and epithelial cells. Intelectin binds to molecules thatcontain sugar residues (such as galF), especially those produced bymicrobes and other non-mammalian organisms (ex. algae, plants).Intelectin is thought to be involved in host-defense as it is producedby epithelial cells facing the environment, such as those lining theairways and gut.

The antibody of the presently disclosed methods is, in some aspects,specific for at least one antigen containing a galactofuranose residueand is selected from the group consisting of monoclonal antibody 205(MAb 205) comprising a variable heavy (VH) domain of SEQ ID NO:1 and avariable light (VL) domain of SEQ ID NO:2; monoclonal antibody 24 (MAb24) comprising a VH domain of SEQ ID NO:3 and a VL domain of SEQ IDNO:4; monoclonal antibody 686 (MAb 686) comprising a VH domain of SEQ IDNO:5 and a VL domain of SEQ ID NO:6; monoclonal antibody 838 (MAb 838)comprising a VH domain of SEQ ID NO:7 and a VL domain of SEQ ID NO:8;and monoclonal antibody 476 (MAb 476) comprising a VH domain of SEQ IDNO:9 and a VL domain of SEQ ID NO:10. The presently disclosed methodsare suitable for use in biological samples selected from the groupconsisting of urine, bronchoalveolar lavage (BAL) fluid, serum,gastrointestinal fluids, blood, saliva and cerebrospinal fluid (CSF).

In other embodiments, the presently disclosed subject matter provides alateral flow device adapted to perform the presently disclosed methodsfor diagnosing a microbial infection in a biological sample of amammalian subject suspected of having, having, or susceptible to havinga microbial infection.

In accordance with some embodiments, presently disclosed subject matterprovides a dipstick assay to perform the presently disclosed methods fordiagnosing a microbial infection in a biological sample of a mammaliansubject suspected of having, having, or susceptible to having amicrobial infection.

In yet other embodiments, the presently disclosed subject matterprovides an antibody specific for at least epitope of an antigensecreted by a microbial organism, wherein in particular aspects, theantigen comprises a galactofuranose residue.

Point-of-Care Diagnostics Such as Lateral Flow Devices and DipstickAssays: Theory and Current Applications

The ability to provide test results rapidly to the patient and/orhealthcare provider is very important to impact outcomes of multipleconditions. Rapid tests to aid diagnosis and enable early detection ofmultiple diseases and physiologic conditions are being developed. Suchtests are especially useful when they can be applied with self-testingand require little in the way of laboratory processing. Examples ofpoint-of-care (POC) test devices in common use today include pregnancyand fertility tests, as well as assays to follow blood glucose indiabetics. Development of diagnostic tests for infections that use POCtesting are especially important in resource-poor settings; for thisreason, POC testing has become a new goal to be achieved for infectionssuch as HIV, malaria, and hepatitis. Similarly, POC testing has thepotential of impacting clinical outcomes when applied to infections thatoccur in the outpatient setting, not only by providing indications ofdisease, but by enabling development of more robust preventionalgorithms.

Commonly used immunoassays in diagnostic and research use includeradio-immunoassays and enzyme-linked immunosorbent assays (ELISAs). Manyof these elaborately configured immunoassays use monoclonal antibodies(mAbs) that possess the ability to bind specifically to the analytebeing tested, thereby enhancing the accuracy of the assay. Variousapproaches have been described for carrying out enzyme immunoassays. Aconsiderable number of these approaches, starting with the earliest ofELISAs, are solid-phase immunoassays in which the analyte to be detectedis bound to a solid matrix directly (Direct ELISA) or indirectly(Sandwich ELISA), in which the analyte is captured on a primary reagent.The choice of the solid matrix depends on procedural considerations. Acommon matrix is the polystyrene surface of multi-well microtiterplates.

These types of assays also are amenable to developing POC devices, inwhich systems can be self-contained so that output is readable by theuser. This characteristic is especially useful when collection of asample to be tested does not require medical intervention (e.g., urine,saliva, or sputum). One device that enables this is the lateral-flowdevice (LFD). These devices use a multi-layered construction containingboth absorbent and non-absorbent components to form a solid-phase. Thecapture and/or recognition reagents (antigen or antibody) arepre-applied to specific areas within the assembled apparatus and theanalyte is allowed to flow through the system to come into contact withreagents. Often, for the purpose of self-containment, the reagentcomponents are added in a dried state so that fluid from the samplere-hydrates and activates them. Conventional ELISA techniques can thenbe used to detect the analyte in the antigen-antibody complex. In someembodiments, the system can be designed to provide a colorimetricreading for visual estimation of a binary response (‘yes’ or ‘no’), orit can be configured to be quantitative.

Lateral flow devices are used to detect analytes in multiple bodyfluids, including serum and urine. To date, these types of devices haveseen the most use for detecting circulating endogenous analytes; perhapsthe most common use of this type of device is in the ubiquitous POCpregnancy test. Current efforts are being directed toward detectingmicrobial analytes, including nucleic acids, in the setting of viralinfections (e.g., influenza, respiratory syncytial virus, and the like),Nielsen, K., et al., Prototype single step lateral flow technology fordetection of avian influenza virus and chicken antibody to avianinfluenza virus. J Immunoassay Immunochem, 2007. 28(4): p. 307-18;Mokkapati, V. K., et al., Evaluation of UPlink-RSV: prototype rapidantigen test for detection of respiratory syncytial virus infection. AnnNY Acad Sci, 2007. 1098: p. 476-85; bacterial infections (e.g., S.pneumoniae, Legionella, Mycobacteria), Koide, M., et al., Comparativeevaluation of Duopath Legionella lateral flow assay against theconventional culture method using Legionella pneumophila and Legionellaanisa strains. Jpn J Infect Dis, 2007. 60(4): p. 214-6.

One assay that is in use worldwide is the BinaxNOW pneumococcal urinaryantigen test; this assay evolved after the serum-based platform wasshown to be effective, but cumbersome. The urinary POC device can beparticularly useful when employed in high-risk patients as a POC testingdevice. Roson, B., et al., Contribution of a urinary antigen assay(Binax NOW) to the early diagnosis of pneumococcal pneumonia. ClinInfect Dis, 2004. 38(2): p. 222-6; Weatherall, C., R. Paoloni, and T.Gottlieb, Point-of-care urinary pneumococcal antigen test in theemergency department for community acquired pneumonia. Emerg Med J,2008. 25(3): p. 144-8. This issue is particularly relevant in thecontext of the presently disclosed subject matter, as thepolysaccharides in the pneumococcus capsule have some structuralsimilarity to those of Aspergillus. Kappe, R. and A. Schulze-Berge, Newcause for false-positive results with the Pastorex Aspergillus antigenlatex agglutination test. J Clin Microbiol, 1993. 31(9): p. 2489-90;Stynen, D., et al., Rat monoclonal antibodies against Aspergillusgalactomannan. Infect Immun, 1992. 60(6): p. 2237-45; Swanink, C. M., etal., Specificity of a sandwich enzyme-linked immunosorbent assay fordetecting Aspergillus galactomannan. J Clin Microbiol, 1997. 35(1): p.257-60.

Lateral Flow Device and Optimized Methods of Use Thereof for DiagnosingMicrobial Infections

Preliminary studies have demonstrated that antigens of A. fumigatus(e.g., galF) are renally concentrated in animal model and are excretedin urine such that the sensitivity and specificity of a urine-basedassay may equal or exceed that of serum based testing. Urinary detectionof antigens would enable development of an easy-to-use POC testingmethod that would enable frequent testing in the outpatient setting,thus aiding the ability to diagnose and optimize screening strategiesemployed to detect infection early in the course of disease.Accordingly, in some embodiments, the presently disclosed subject matterprovides a POC test to detect Aspergillus galF-containing antigens inurine. Monoclonal antibodies that recognize galactofuranose residues ofA. fumigatus galF have been developed and are used in the presentlydisclosed galF test.

A standard ELISA format was used as a screen to identify antibodies touse for capture on the immobilized device. The identified antibody canbe used as a capture antibody with point of care testing device (strip),which can be optimized for conditions to detect galF-antigen (antibodyconcentration, incubation conditions, and the like).

The term “dipstick assay” as used herein means any assay using adipstick in which sample solution is contacted with the dipstick tocause sample solution to move by capillary action to a capture zone ofthe dipstick thereby allowing a target antigen in the sample solution tobe captured and detected at the capture zone. To test for the presenceof analyte, the contact end of the dipstick is contacted with the testsolution. If analyte is present in the test solution it travels to thecapture zone of the dipstick by capillary action where it is captured bythe capture antibody. The presence of analyte at the capture zone of thedipstick is detected by a further anti-analyte antibody (the detectionantibody) labelled with, for example, colloidal gold.

These dipstick tests have several advantages. They are easy and cheap toperform, no specialist instruments are required, and the results areobtained rapidly and can be read visually. These tests are, therefore,particularly suited for use in a physician's office, at home, in remoteareas, and in developing countries where specialist equipment may not beavailable. They can be used, for example, to test whether a patient isinfected with a disease causing microorganism such as A. fumigatus.

To perform a method of the first aspect of the invention, the targetingagent and labels may simply be added to the test solution and the testsolution then contacted with the contact end of the chromatographicstrip. Such methods are easier to perform than the method disclosed inWO 00/25135 in which two separate wicking steps are required. Theresults may, therefore, be obtained more rapidly, and yet thesensitivity of analyte detection is higher.

The term “chromatographic strip” is used herein to mean any porous stripof material capable of transporting a solution by capillarity. Thechromatographic strip may be capable of bibulous or non-bibulous lateralflow, but preferably bibulous lateral flow. By the term “non-bibulouslateral flow” is meant liquid flow in which all of the dissolved ordispersed components of the liquid are carried at substantially equalrates and with relatively unimpaired flow laterally through the membraneas opposed to preferential retention of one or more components as wouldoccur with “bibulous lateral flow.” Materials capable of bibulouslateral flow include paper, nitrocellulose, and nylon. A preferredexample is nitrocellulose.

The labels may be bound to the ligands of the targeting agent bypre-mixing the targeting agent with the labels before the targetingagent is added to (or otherwise contacted with) the test solution.However, in some circumstances, it is preferred that the targeting agentand labels are not pre-mixed because such pre-mixing can cause thetargeting agent and labels to precipitate. Thus, the targeting agent andthe labels may be added separately to (or contacted separately with) thetest solution. The targeting agent and the labels can be added to (orcontacted with) the test solution at substantially the same time, or inany order.

The test solution may be pre-incubated with the targeting agent andlabels before the test solution is contacted with the contact end of thechromatographic strip to ensure complex formation. The optimal time ofpre-incubation will depend on the ratio of the reagents and the flowrate of the chromatographic strip. In some cases, pre-incubation for toolong can decrease the detection signal obtained, and even lead to falsepositive detection signals. Thus, it may be necessary to optimize thepre-incubation time for the particular conditions used.

It may be desired to pre-incubate the targeting agent with the testsolution before binding the labels to the targeting agent so that thetargeting agent can be allowed to bind to analyte in the test solutionunder optimum binding conditions. Generally, the presently disclosedsubject matter provides a method for diagnosing a microbial infection ina biological sample from a mammalian subject suspected of having,having, or susceptible to having a microbial infection, by detecting thepresence of at least one antigen comprising a galF residue in abiological sample of the mammalian subject, the method comprising: (a)treating the biological sample to decrease or minimize human IntL-1binding of galF residues present in the sample; (b) contacting thetreated sample of (a) with at least one antibody specific for at leastone antigen comprising a galF residue in an effective amount to producea detectable amount of antibody-galF antigen complex; and (c) detectingthe presence of at least one antibody-galF antigen complex, wherein thedetection of the presence of at least one antibody-galF antigen complexis diagnostic of a microbial infection in a mammalian subject.

In accordance with another embodiment, the present invention provides amethod for diagnosing a microbial infection in a biological sample froma mammalian subject suspected of having, having, or susceptible tohaving a microbial infection by detecting the presence of at least oneantigen comprising a galF residue in a biological sample of themammalian subject, the method comprising: (a) treating the biologicalsample comprising contacting the sample with a substrate such as aligand which binds directly to intelectin, or calcium or mono anddivalent cations with high affinity, to inhibit hIntL binding of galFresidues present in the sample; (b) contacting the treated sample of (a)with at least one antibody specific for at least one antigen comprisinga galF residue in an effective amount to produce a detectable amount ofantibody-galF antigen complex; and (c) detecting the presence of atleast one antibody-galF antigen complex, wherein the detection of thepresence of at least one antibody-galF antigen complex is diagnostic ofa microbial infection in a mammalian subject.

The microbial infection can be selected from the group consisting of abacterial infection and a fungal infection. In some embodiments, thebacterial infection is caused by an infection of Streptococcuspneumoniae. In other embodiments the microbial infection is a fungalinfection caused by an infection of an organism selected from the groupconsisting of Aspergillus species, Fusarium species, Coccidiodesspecies, Cryptococcus species, and Histoplasma species.

In some embodiments, the microbial infection is caused by Prokaryoticand Eukaryotic pathogens that produce galF-containing antigens,including but not limited to, Streptococcus species, Pseudomonasspecies, Nocardia species, Actinomyces species. Zygomycetes andparasites, including but not limited to Leishmania species, andTrypanosoma species.

In particular embodiments, at least one antibody specific for at leastone antigen comprising a galactofuranose residue is selected from thegroup consisting of monoclonal antibody 205 (MAb 205) comprising avariable heavy (VH) domain of SEQ ID NO:1 and a variable light (VL)domain of SEQ ID NO:2; monoclonal antibody 24 (MAb 24) comprising a VHdomain of SEQ ID NO:3 and a VL domain of SEQ ID NO:4; monoclonalantibody 686 (MAb 686) comprising a VH domain of SEQ ID NO:5 and a VLdomain of SEQ ID NO:6; monoclonal antibody 838 (MAb 838) comprising a VHdomain of SEQ ID NO:7 and a VL domain of SEQ ID NO:8; and monoclonalantibody 476 (MAb 476) comprising a VH domain of SEQ ID NO:9 and a VLdomain of SEQ ID NO:10.

One of ordinary skill in the art upon review of the presently disclosedsubject matter would appreciate that any biological fluid in which atleast one antigen comprising a galactofuranose residue is secreted issuitable for use with the presently disclosed methods. In particularembodiments, the biological sample is selected from the group consistingof urine, bronchoalveolar lavage (BAL) fluid, serum, gastrointestinalfluids, blood, and cerebrospinal fluid (CSF).

In some embodiments, the presently disclosed methods further comprisepre-treating the biological sample before contacting the biologicalsample with at least one antibody specific for at least one antigencomprising a galactofuranose residue. The pre-treating step can includea step selected from the group consisting of filtering, diluting, andconcentrating the biological sample, and combinations thereof.

Without being held to any particular theory, the Mab476 antibody used inthe methods of the present invention is thought to bind to thegalF-containing O-glycan moiety/moieties associated with cellulose(CelA) protein. As described above, the Mab476 antibody may bind to galFfrom any origin, including galF that is present on extracellularvesicles shed by the infectious organism.

In accordance with some embodiments, the method for treating the samplein step (a) comprises contacting the sample with a substrate which bindsCa²⁺ ions with high affinity.

Examples of substrates which can bind divalent cations with highaffinity include, for example,N,N,N′,N′-tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN,membrane-permeable chelator) and diethylenetriaminepentaacetic acid(DTPA, membrane-impermeable chelator), and cation exchange resins suchas AG50, Chelex, poly(acrylic acid).

In accordance with some embodiments, the method for treating the samplein step (a) comprises contacting the sample with a compound whichchelates Ca²⁺ ions with high affinity. Examples of chelators include,without limitation, ethylenediamine tetraacetic acid (EDTA), Ethyleneglycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA),1,2-bis(o-Aminophenoxy)ethane-N,N,N′,N′-tetraacetic Acid (BAPTA),1-(2-Nitro-4,5-dimethoxyphenyl)-1,2-diaminoethane-N,N,N′,N′-tetraaceticAcid, 4Na, Dimethoxynitrophenamine (DM-Nitrophen), and others.

In accordance with some embodiments, the method for treating the samplein step (a) comprises contacting the sample with EDTA and/or EGTA.

In accordance with some embodiments, the method for treating the samplein step (a) comprises contacting the sample with a substrate which bindshIntL-1 with high affinity.

Examples of compounds which bind hIntL-1 include, but are not limitedto, glycerol, 3-keto-2-deoxyoctonic acid, D-glycerol-1-phosphate,D-mannoheptose, and other compounds which are bound by hIntL-1.

In accordance with some embodiments, the method for treating the samplein step (a) comprises contacting the sample with an antibody specificfor hIntL-1. In some embodiments, the antibody can be rabbit polyclonalIgG anti-hIntL-1 antibody.

In accordance with some embodiments, the method for treating the samplein step (a) comprises contacting the sample with one or more compoundswhich are bound by hIntL-1 with high affinity.

In accordance with some embodiments, the method for treating the samplein step (a) comprises contacting the sample with one or more compoundswhich bind hIntL with high affinity selected from the group consistingof glycerol, 3-Keto-2-deoxyoctonic acid; D-glycerol-1-phosphate,D-mannoheptose, sepharose, sepharose-containing particles (i.e. latex,polystyrene or glass beads, microspheres or gels).

In accordance with some embodiments, the method for treating the samplein step (a) comprises a combination of one or more of the above methodsincluding, for example, treating the sample with a chelator and one ormore compounds which are bound by hIntL with high affinity, and ananti-IntL antibody. Any of the above methods can be combined to furtherprevent hIntL-1 from binding galF in a biological sample.

In accordance with some embodiments, the method for treating the samplein step (a) comprises contacting the sample with a desalting column.Examples of desalting columns are known in the art, including, forexample, desalting columns which are pre-packed with polyacrylamide sizeexclusion resins.

The subject treated by the presently disclosed methods in their manyembodiments is desirably a human subject, although it is to beunderstood that the methods described herein are effective with respectto all vertebrate species, which are intended to be included in the term“subject.” Accordingly, a “subject” can include a human subject formedical purposes, such as for the treatment of an existing condition ordisease or the prophylactic treatment for preventing the onset of acondition or disease, or an animal subject for medical, veterinarypurposes, or developmental purposes. Suitable animal subjects includemammals including, but not limited to, primates, e.g., humans, monkeys,apes, and the like; bovines, e.g., cattle, oxen, and the like; ovines,e.g., sheep and the like; caprines, e.g., goats and the like; porcines,e.g., pigs, hogs, and the like; equines, e.g., horses, donkeys, zebras,and the like; felines, including wild and domestic cats; canines,including dogs; lagomorphs, including rabbits, hares, and the like; androdents, including mice, rats, and the like. An animal may be atransgenic animal. In some embodiments, the subject is a humanincluding, but not limited to, fetal, neonatal, infant, juvenile, andadult subjects. Further, a “subject” can include a patient afflictedwith or suspected of being afflicted with a condition or disease. Thus,the terms “subject” and “patient” are used interchangeably herein. Inparticular embodiments, the subject is a human adult suspected ofhaving, having, or susceptible of having a microbial infection. In otherembodiments, the subject is a human child, e.g., a human less than about19 years of age, suspected of having, having, or susceptible of having amicrobial infection.

The presently disclosed methods can be used to diagnose, for theprognosis, or the monitoring of a disease state or condition. As usedherein, the term “diagnosis” refers to a predictive process in which thepresence, absence, severity or course of treatment of a disease,disorder or other medical condition is assessed. For purposes herein,diagnosis also includes predictive processes for determining the outcomeresulting from a treatment. Likewise, the term “diagnosing,” refers tothe determination of whether a sample specimen exhibits one or morecharacteristics of a condition or disease. The term “diagnosing”includes establishing the presence or absence of, for example, a targetantigen or reagent bound targets, or establishing, or otherwisedetermining one or more characteristics of a condition or disease,including type, grade, stage, or similar conditions. As used herein, theterm “diagnosing” can include distinguishing one form of a disease fromanother. The term “diagnosing” encompasses the initial diagnosis ordetection, prognosis, and monitoring of a condition or disease.

The term “prognosis,” and derivations thereof, refers to thedetermination or prediction of the course of a disease or condition. Thecourse of a disease or condition can be determined, for example, basedon life expectancy or quality of life. “Prognosis” includes thedetermination of the time course of a disease or condition, with orwithout a treatment or treatments. In the instance where treatment(s)are contemplated, the prognosis includes determining the efficacy of atreatment for a disease or condition.

As used herein, the term “risk” refers to a predictive process in whichthe probability of a particular outcome is assessed. The term“monitoring,” such as in “monitoring the course of a disease orcondition,” refers to the ongoing diagnosis of samples obtained from asubject having or suspected of having a disease or condition. The term“marker” refers to a molecule, including an antigen, such as apolysaccharide, that when detected in a sample is characteristic of orindicates the presence of a disease or condition.

Accordingly, in some embodiments, the presently disclosed subject matterprovides a method for diagnosing of a microbial infection in a mammaliansubject suspected of having, having, or susceptible to having amicrobial infection, wherein the method comprises monitoring a treatmentregimen of a microbial infection to determine the efficacy of thetreatment regimen.

In accordance with some embodiments, the methods disclosed herein can beused with lateral flow devices such as those disclosed in in U.S. patentapplication Ser. No. 13/511,264, and incorporated by reference herein inits entirety. The presently disclosed methods can use a lateral flowdevice or dipstick assay comprising an immunochromatographic strip testthat relies on a direct (double antibody sandwich) reaction. Withoutwishing to be bound to any one particular theory, this direct reactionscheme is best used when sampling for larger analytes that may havemultiple antigenic sites. Different antibody combinations can be used,for example different antibodies can be included on the capture(detection) line, the control line, and included in the mobile phase ofthe assay, for example, as conjugated to gold particles, e.g., goldmicroparticles or gold nanoparticles.

In an embodiment, the present disclosure comprises kits for diagnosing amicrobial infection in a biological sample from a mammalian subjectsuspected of having, having, or susceptible to having a microbialinfection by detecting the presence of at least one antigen comprising agalactofuranose residue in a biological sample of the mammalian subject.Such kits may include all necessary reagents, components, apparatus andinstructions for treating the biological sample to inhibit humanintelectin (hIntL) binding of galactofuranose residues present in thesample; in an embodiment, the kits may further comprise at least oneantibody specific for at least one antigen comprising a galactofuranoseresidue in an effective amount to produce a detectable amount ofantibody-galF antigen complex; in an embodiment, the kit further enablesdetecting the presence of at least one antibody-galF antigen complex,wherein the detection of the presence of at least one antibody-galFantigen complex is diagnostic of a microbial infection in a mammaliansubject. In certain embodiments, the kit comprises the use of a lateralflow apparatus, dipstick, assay stick with immunochromatographicdetection display, and any such apparatus know to those skilled in theart. In certain embodiments, reagents and/or detection components may beimmobilized on the apparatus itself (i.e. on the dipstick). In certainembodiments, reagents for chelating calcium are included in the kit.

As used herein the term “lateral flow” refers to liquid flow along theplane of a substrate or carrier, e.g., a lateral flow membrane. Ingeneral, lateral flow devices comprise a strip (or a plurality of stripsin fluid communication) of material capable of transporting a solutionby capillary action, i.e., a wicking or chromatographic action, whereindifferent areas or zones in the strip(s) contain assay reagents, whichare either diffusively or non-diffusively bound to the substrate, thatproduce a detectable signal as the solution is transported to ormigrates through such zones. Typically, such assays comprise anapplication zone adapted to receive a liquid sample, a reagent zonespaced laterally from and in fluid communication with the applicationzone, and a detection zone spaced laterally from and in fluidcommunication with the reagent zone. The reagent zone can comprise acompound that is mobile in the liquid and capable of interacting with ananalyte in the sample, e.g., to form an analyte-reagent complex, and/orwith a molecule bound in the detection zone. The detection zone maycomprise a binding molecule that is immobilized on the strip and iscapable of interacting with the analyte and/or the reagent and/or ananalyte-reagent complex to produce a detectable signal. Such assays canbe used to detect an analyte in a sample through direct (sandwich assay)or competitive binding. Examples of lateral flow devices are provided inU.S. Pat. No. 6,194,220 to Malick et al.; U.S. Pat. No. 5,998,221 toMalick et al.; U.S. Pat. No. 5,798,273 to Shuler et al.; and RE38,430 toRosenstein.

In some embodiments, the presently disclosed methods can be used with anassay comprising a sandwich lateral flow or dipstick assay. In asandwich assay, a liquid sample that may or may not contain an analyteof interest is applied to the application zone and allowed to pass intothe reagent zone by capillary action. The term “analyte” as used hereinrefers to an antigen comprising a galactofuranose residue. In certainembodiments the presence or absence of an analyte in a sample isdetermined qualitatively. In other embodiments, a quantitativedetermination of the amount or concentration of analyte in the sample isdetermined.

The analyte, if present, interacts with a labeled reagent in the reagentzone to form an analyte-reagent complex and the analyte-reagent complexmoves by capillary action to the detection zone. The analyte-reagentcomplex becomes trapped in the detection zone by interacting with abinding molecule specific for the analyte and/or reagent. Unbound samplecan pass through the detection zone by capillary action to a controlzone or an absorbent pad laterally juxtaposed and in fluid communicationwith the detection zone. The labeled reagent may then be detected in thedetection zone by appropriate means.

Generally, and without limitation, lateral flow devices comprise asample pad. A sample pad comprises a membrane surface, also referred toherein as a “sample application zone,” adapted to receive a liquidsample. A standard cellulose sample pad has been shown to facilitateabsorption and flow of biological samples, including, but not limitedto, urine. The sample pad comprises a portion of lateral flow devicethat is in direct contact with the liquid sample, that is, it receivesthe sample to be tested for the analyte of interest. The sample pad canbe part of, or separate from, a lateral flow membrane. Accordingly, theliquid sample can migrate, through lateral or capillary flow, fromsample pad toward a portion of the lateral flow membrane comprising adetection zone. The sample pad is in fluid communication with thelateral flow membrane comprising an analyte detection zone. This fluidcommunication can arise through either be an overlap, top-to-bottom, oran end-to-end fluid connection between the sample pad and a lateral flowmembrane. In certain embodiments, the sample pad comprises a porousmaterial, for example and not limited to, paper. In certain embodimentsthe targeting agent, molecule or other reagent of the diagnostic methodmay be immobilized on the conjugate pad. In certain embodiments, thetargeting agent, molecule or other reagent of the diagnostic method maybe present in an alternative format.

The term “sample” as used herein refers to any biological samplesuspected of containing an analyte for detection or a control sampleexpected to be substantially free of the analyte of interest. Inparticular embodiments, the sample comprises a biological fluid of asubject suspected of having, having, or susceptible of having amicrobial infection. In some embodiments, the biological sample is inliquid form, while in other embodiments it can be changed into a liquidform, e.g., by reconstitution in a suitable solvent, e.g., an aqueoussolution. The presently disclosed lateral flow devices are suitable foruse with a variety of biological samples including, but not limited to,urine, bronchoalveolar lavage (BAL) fluid, serum, blood,gastrointestinal fluids, and cerebrospinal fluid (CSF).

Typically, a sample pad is positioned adjacent to and in fluidcommunication with a conjugate pad. A conjugate pad comprises a labeledreagent having specificity for one or more analytes of interest. In someembodiments, the conjugate pad comprises a non-absorbent, syntheticmaterial (e.g., polyester) to ensure release of its contents. Adetection conjugate is dried into place on the conjugate pad and onlyreleased when the liquid sample is applied to the sample pad. Detectionconjugate can be added to the pad by immersion or spraying.

In particular embodiments, the detection conjugate comprises an antibodyhaving specificity for a antigen comprising a galactofuranose residue.In representative embodiments, the antibody is selected from the groupconsisting of monoclonal antibody 205 (MAb 205) comprising a variableheavy (VH) domain of SEQ ID NO:1 and a variable light (VL) domain of SEQID NO:2; monoclonal antibody 24 (MAb 24) comprising a VH domain of SEQID NO:3 and a VL domain of SEQ ID NO:4; monoclonal antibody 686 (MAb686) comprising a VH domain of SEQ ID NO:5 and a VL domain of SEQ IDNO:6; monoclonal antibody 838 (MAb 838) comprising a VH domain of SEQ IDNO:7 and a VL domain of SEQ ID NO:8; and monoclonal antibody 476 (MAb476) comprising a VH domain of SEQ ID NO:9 and a VL domain of SEQ IDNO:10. The antibody, e.g., a monoclonal antibody (MAb), can beconjugated to a gold particle, e.g., colloidal gold, including goldmicrospheres, or gold nanoparticles. For example, it is possible tobiotinylate the conjugated MAb to take advantage of the strong affinitythat biotin has for streptavidin, using Streptavidin-coatedmicrospheres. Alternatives include protein A-coated microspheres thatbind to Fc region of IgGs. Conditions to define optimal optimization tocolloidal gold can be determined, for example, in microtiter wells. Forexample, 100 μL of colloidal gold at 1 OD530 can be added to each well,followed by 10 μL of 22 mM buffers (MES, HEPES) at variable pH (5.5 to10, in 0.5 increments). Antibodies can be added at concentrationsranging from about 1.25 μg/l OD colloid to about 10 μg/l OD colloid,incubated for 15 minutes, and then 25 μL of 1.5 NaCl can be added.Conjugated particles will be stable and pink; the optimal condition thatrequires the lowest concentration of antibodies can be determined.

It is contemplated that any detection agent used in the presentdisclosure will be labeled with any “reporter molecule,” so that it isdetectable in any detection system, including, but not limited to enzyme(e.g., ELISA, as well as enzyme-based histochemical assays),fluorescent, radioactive, and luminescent systems. It is not intendedthat the present invention be limited to any particular detection systemor label. Furthermore, it is contemplated that screening for the targetwill be accomplished by techniques known in the art (e.g.,radioimmunoassay, ELISA (enzyme-linked immunosorbant assay), “sandwich”immunoassays, immunoradiometric assays, gel diffusion precipitationreactions, immuNIPA-liffusion assays, in situ immunoassays (e.g., usingcolloidal gold, enzyme or radioisotope labels), Western blots,precipitation reactions, agglutination assays (e.g., gel agglutinationassays, hemagglutination assays, etc.), complement fixation assays,immunofluorescence assays, color emitting nanoparticles (e.g. quantumdots), protein A assays, and immunoelectrophoresis assays, etc. Suchmethodologies and techniques are known to those skilled in the art.

Usually, the conjugate pad is adjacent to and in fluid communicationwith a lateral flow membrane. Capillary action draws a fluid mixture upthe sample pad, through the conjugate pad where an antibody-galF antigencomplex is formed, and into the lateral flow membrane. Lateral flow is afunction of the properties of the lateral flow membrane. The lateralflow membrane typically is extremely thin and is hydrophilic enough tobe wetted, thereby permitting unimpeded lateral flow and mixture ofreactants and analytes at essentially the same rates.

Lateral flow membranes can comprise any substrate capable of providingliquid flow including, but not limited to, substrates, such asnitrocellulose, nitrocellulose blends with polyester or cellulose,untreated paper, porous paper, rayon, glass fiber, acrylonitrilecopolymer, plastic, glass, or nylon. Lateral flow membranes can beporous. Typically, the pores of a lateral flow membrane are ofsufficient size such that particles, e.g., microparticles comprising areagent capable of forming a complex with an analyte, flow through theentirety of the membrane. Lateral flow membranes, in general, can have apore size ranging from about 3 μm to about 100 μm, and, in someembodiments, have a pore size ranging from about 10 μm to about 50 μm.Pore size affects capillary flow rate and the overall performance of thedevice.

There are multiple benefits to using nitrocellulose for the primarymembrane: low cost, capillary flow, high affinity for protein biding,and ease of handling. Nitrocellulose has high protein binding. Anotheralternative is cellulose acetate, which has low protein binding. Sizedictating surface area dictates membrane capacity (the volume of samplethat can pass through the membrane per unittime=length×width×thickness×porosity. Because these variables controlthe rate at which lateral flow occurs, they can impact sensitivity andspecificity of the assay. The flow rate also varies with sampleviscosity. Several different sizes and polymers are available for use asmicrospheres, which migrate down the membrane with introduction of thefluidic sample. The optimal flow rate generally is achieved usingspheres that are 1/10 the pore size of the membrane or smaller.

One skilled in the art will be aware of other materials that allowliquid flow. Lateral flow membranes, in some embodiments, can compriseone or more substrates in fluid communication. For example, a conjugatepad can be present on the same substrate or may be present on separatesubstrates (i.e., pads) within or in fluid communication with lateralflow membranes. In some embodiments, the nitrocellulose membrane cancomprise a very thin Mylar sheet coated with a nitrocellulose layer.

Lateral flow membranes can further comprise at least one indicator zoneor detection zone. The terms “indicator zone” and “detection zone” areused interchangeably herein and mean the portion of the carrier orporous membrane comprising an immobilized binding reagent. As usedherein, the term “binding reagent” means any molecule or a moleculebound to a particle, wherein the molecule recognizes or binds theanalyte in question. The binding reagent is capable of forming a bindingcomplex with the analyte-labeled reagent complex. The binding reagent isimmobilized in the detection zone and is not affected by the lateralflow of the liquid sample due to the immobilization on the membrane.Once the binding reagent binds the analyte-labeled reagent complex itprevents the analyte-labeled reagent complex from continuing with theflow of the liquid sample. In some embodiments, the binding reagent isan antibody having specificity for an antigen having at least onegalactofuranose residue.

Accordingly, during the actual reaction between the analyte and thereagent, the first member binds in the indicator zone to the secondmember and the resulting bound complex is detected with specificantibodies. Detection may use any of a variety of labels and/or markers,e.g., enzymes (alkaline phosphatase or horseradish peroxidase withappropriate substrates), radioisotopes, liposomes or latex beadsimpregnated with fluorescent tags, polymer dyes or colorednanoparticles, and the like. Thus, the result can be interpreted by anydirect or indirect reaction. Colloidal gold particles, which impart apurple or red coloration, are most commonly used currently.

The capture and immobilization of the assay reagent (complementarymember of the binding pair) at the indicator zone can be accomplished bycovalent bonding or, more commonly, by adsorption, such as by drying.Such capture also can be indirect, for example, by binding of latexbeads coated with the reagent. Depending on the nature of the materialcomprising the lateral flow membrane, covalent bonding may be enabled,for example with use of glutaraldehyde or a carbodiimide. Inimmunoassays, most common binding pairs are antigen-antibody pairs;however, multiple other binding pairs can be performed, such asenzyme-substrate and receptor-ligand.

In some embodiments, the indicator zone further comprises a test lineand a control line. A test line can comprise an immobilized bindingreagent. When antibodies are used to develop a test line in the LFD thatemploys a sandwich type of assay, they are applied at a ratio of about1-3 μg/cm across the width of a strip 1 mm wide; hence, antibodyconcentration is about 10-30 μg/cm², which is about 25-100 fold thatused in an ELISA. Brown, M. C., Antibodies: key to a robust lateral flowimmunoassay, in Lateral Flow Immunoassay, H. Y. T. R. C. Wong, Editor.2009, Humana Press: New York, N.Y. p. 59-74.

Further, in some embodiments, the presently disclosed lateral flowassays can be used to detect multiple analytes in a sample. For example,in a lateral flow assay, the reagent zone can comprise multiple labeledreagents, each capable of binding to a different analyte in a liquidsample or a single labeled reagent capable of binding to multipleanalytes. If multiple labeled reagents are used in a lateral flow assay,the reagents may be differentially labeled to distinguish differenttypes of analytes in a liquid sample.

It also is possible to place multiple lines of capture antibodies on themembrane to detect different analytes. Combinations of antibodies thatdetect different epitopes of glycans may optimize specificity if it isfound that one antibody performs at a low quantitative limit ofdetection, yet exhibits some degree of nonspecific binding (or bindingto urine components in control animals). One possibility is that thedevice may be adapted to detect galF and another fungal component toincrease the potential spectrum of pathogens detected and to increasespecificity of the reaction. Aspergillus species are thought to secretegalF and other fungal components, while glycans from other‘contaminants’ should not contain other fungal components.

For quality control, typically a lateral flow membrane can include acontrol zone comprising a control line. The term “control zone” refersto a portion of the test device comprising a binding molecule configuredto capture the labeled reagent. In a lateral flow assay, the controlzone may be in liquid flow contact with the detection zone of thecarrier, such that the labeled reagent is captured on the control lineas the liquid sample is transported out of the detection zone bycapillary action. Detection of the labeled reagent on the control lineconfirms that the assay is functioning for its intended purpose.Placement of a control line can be accomplished using a microprocessorcontrolled TLC spotter, in which a dispenser pump releases a constantvolume of reagent across the membrane.

A typical lateral flow device can also comprises an absorbent pad. Theabsorbent pad comprises an “absorbent material,” which as used herein,refers to a porous material having an absorbing capacity sufficient toabsorb substantially all the liquids of the assay reagents and any washsolutions and, optionally, to initiate capillary action and draw theassay liquids through the test device. Suitable absorbent materialsinclude, for example, nitrocellulose, nitrocellulose blends withpolyester or cellulose, untreated paper, porous paper, rayon, glassfiber, acrylonitrile copolymer, plastic, glass, or nylon.

In some embodiments, a lateral flow membrane is bound to one or moresubstantially fluid-impervious sheets, one on either side, e.g., abottom sheet and a complimentary top sheet with one or more windowsdefining an application zone and an indicator zone.

A typical lateral flow device also can include a housing. The term“housing” refers to any suitable enclosure for the presently disclosedlateral flow devices. Exemplary housings will be known to those skilledin the art. The housing can have, for example, a base portion and a lidportion. The lid portion can include a top wall and a substantiallyvertical side wall. A rim may project upwardly from the top wall and mayfurther define a recess adapted to collect a sample from a subject.Suitable housings include those provided in U.S. Pat. No. 7,052,831 toFletcher et al and those used in the BD Directigen™ EZ RSV lateral flowassay device.

As with the general method described immediately hereinabove, themicrobial infection can be selected from the group consisting of abacterial infection and a fungal infection. In some embodiments, thebacterial infection is caused by an infection of Streptococcuspneumoniae. In particular embodiments, the microbial infection is afungal infection caused by an infection of an organism selected from thegroup consisting Aspergillus species, Fusarium species, Coccidioidesspecies, Cryptococcus species, Histoplasma species and Zygomycetes.

In some embodiments, the microbial infection is caused by an organismhaving a propensity to cause lung infection, including but not limitedto, Streptococcus species, Pseudomonas species, Nocardia species,Actinomyces species, Mycobacteria species as well as fungal organismssuch as Aspergillus species, Cryptococcus species, Histoplasma species,and Zygomycetes.

In some embodiments, an antigen having a galactofuranose residue can bemeasured in whole, unconcentrated, or otherwise unprocessed, biologicalsamples using the presently disclosed methods and devices. In otherembodiments, the biological sample can be processed, e.g., concentrated,diluted, filtered, and the like, prior to performing the test. Thepre-treatment of the urine sample can include diluting the urine samplein an aqueous solution, concentrating the urine sample, filtering theurine sample, or a combination thereof.

One of ordinary skill in the art upon review of the presently disclosedsubject matter would appreciate that the pre-treatment steps can beperformed in any particular order, e.g., in some embodiments, the samplecan be diluted or concentrated and then filtered, whereas in otherembodiments, the sample can be filtered and then diluted orconcentrated. In particular embodiments, the presently disclosed methodsinclude filtering the urine sample, for example, through a desaltingcolumn, to remove an inhibitor that interferes with the detection ofantigen in the urine sample. This step can be performed with or withoutany further dilution or concentration of the sample.

Thus, in some embodiments, the lateral flow device further comprises anapparatus adapted to pre-treat the biological sample before contactingthe biological sample with at least one antibody specific for at leastone polysaccharide comprising a galF residue. In particular embodiments,the apparatus is adapted to filter, dilute, or concentrate thebiological sample, or combinations thereof. More particularly, theapparatus can be adapted to remove an inhibitor that interferes with thedetection of the at least one antigen comprising a galF residue in thebiological sample, in particular, a urine sample.

In other embodiments, different parameters of the test, e.g., incubationtime, can be manipulated to increase sensitivity and/or specificity ofthe test to eliminate the need for processing the biological sample.Accordingly, in some embodiments, the presently disclosed subject matterprovides an antibody specific for at least epitope of an antigensecreted by a microbial organism. In particular embodiments, the antigencomprises a galF residue. In more particular embodiments, the antibodyis specific for at least one epitope of an antigen secreted by amicrobial organism selected from the group consisting of Aspergillusspecies, Fusarium species, Coccidioides species, Cryptococcus species,Histoplasma species, Zygomycetes and certain Streptococcus species. Inadditional embodiments, the antibody is specific for at least oneepitope of a polysaccharide secreted by a microbial organism selectedfrom the group consisting of Streptococcus species, Pseudomonas species,Nocardia species, Actinomyces species, Mycobacteria species, Leishmaniaspecies and Trypanosoma species

Also provided herein are kits comprising components of a diagnosticregimen, for example components for processing a sample along with adetection assay, lateral flow device, dipstick, and instructions forusing the same. The kit can also comprise packaging or a containerhousing at least one or more components of the diagnostic assay, and canalso comprise instructions on storage, administration, dosing or thelike and/or an insert regarding the active ingredients. The kit can alsocomprise instructions for monitoring the presence and/or prevalence ofan infectious organisms (or metabolites thereof) once administered, andoptionally, materials for performing such assays including, e.g.,reagents, well plates, containers, markers or labels, spin columns, andthe like. Other suitable components to include in kits of the disclosurewill be readily apparent to one of skill in the art, taking intoconsideration the infectious organism to be detected, sample to beprocessed, and storage conditions.

Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation. Unlessotherwise defined, all technical and scientific terms used herein havethe same meaning as commonly understood by one of ordinary skill in theart to which this presently described subject matter belongs.

Following long-standing patent law convention, the terms “a,” “an,” and“the” refer to “one or more” when used in this application, includingthe claims. Thus, for example, reference to “a subject” includes aplurality of subjects, unless the context clearly is to the contrary(e.g., a plurality of subjects), and so forth.

Throughout this specification and the claims, the terms “comprise,”“comprises,” and “comprising” are used in a non-exclusive sense, exceptwhere the context requires otherwise. Likewise, the term “include” andits grammatical variants are intended to be non-limiting, such thatrecitation of items in a list is not to the exclusion of other likeitems that can be substituted or added to the listed items.

For the purposes of this specification and appended claims, unlessotherwise indicated, all numbers expressing amounts, sizes, dimensions,proportions, shapes, formulations, parameters, percentages, parameters,quantities, characteristics, and other numerical values used in thespecification and claims, are to be understood as being modified in allinstances by the term “about” even though the term “about” may notexpressly appear with the value, amount or range. Accordingly, unlessindicated to the contrary, the numerical parameters set forth in thefollowing specification and attached claims are not and need not beexact, but may be approximate and/or larger or smaller as desired,reflecting tolerances, conversion factors, rounding off, measurementerror and the like, and other factors known to those of skill in the artdepending on the desired properties sought to be obtained by thepresently disclosed subject matter. For example, the term “about,” whenreferring to a value can be meant to encompass variations of, in someembodiments, ±100% in some embodiments ±50%, in some embodiments ±20%,in some embodiments ±10%, in some embodiments ±5%, in some embodiments±1%, in some embodiments ±0.5%, and in some embodiments ±0.1% from thespecified amount, as such variations are appropriate to perform thedisclosed methods or employ the disclosed compositions.

Further, the term “about” when used in connection with one or morenumbers or numerical ranges, should be understood to refer to all suchnumbers, including all numbers in a range and modifies that range byextending the boundaries above and below the numerical values set forth.The recitation of numerical ranges by endpoints includes all numbers,e.g., whole integers, including fractions thereof, subsumed within thatrange (for example, the recitation of 1 to 5 includes 1, 2, 3, 4, and 5,as well as fractions thereof, e.g., 1.5, 2.25, 3.75, 4.1, and the like)and any range within that range.

EXAMPLES

The following examples have been included to provide guidance to one ofordinary skill in the art for practicing representative embodiments ofthe presently disclosed subject matter. In light of the presentdisclosure and the general level of skill in the art, those of skill canappreciate that the following examples are intended to be exemplary onlyand that numerous changes, modifications, and alterations can beemployed without departing from the scope of the presently disclosedsubject matter. The synthetic descriptions and specific examples thatfollow are only intended for the purposes of illustration, and are notto be construed as limiting in any manner to make compounds of thedisclosure by other methods.

Example 1

Recombinant IntL-1 Binds to Aspergillus Ethanol Precipitate and WholeCells (Conidia and Hyphae).

Method for Ethanol Precipitate binding.

Wells of a microtiter plate were coated with 10 μg/ml of EP antigen andblocked with a blocking buffer containing 0.1% BSA in IntL-ELISA buffer(20 mM HEPES pH7.4; 150 mM NaCl; 10 mM CaCl2; 0.1% Tween-20. Ref:Wesener et al. (2015) Nature Structural & Molecular Biology, 22(8):603).Recombinant human IntL-1 (rIntL-1) was serially diluted tenfold inblocking buffer starting at 10⁻⁶ M, and incubated on the EP-coated platefor 2 h at 30° C., followed by washes with IntL-ELISA buffer. The boundIntL was detected by an anti-omentin Rabbit IgG ab (Millipore) diluted1:1000 in blocking buffer and incubated at 30° C. for 2 h. Wells wereagain washed and incubated with a goat anti-Rabbit IgG-AP at 1:1000 for1 h at 30° C. Following another wash, the color was developed by APsubstrate for 30 minutes at 37° C.

Method for Conidia & Hyphae Binding.

Conidia from Aspergillus fumigatus expressing a green fluorescentprotein (GFP) were counted and adjusted to 10⁶/mL in Sabouraud'sDextrose broth, and grown for 6 hours at 37° C. to germinate (forminghyphae). The collection of swollen, germinating conidia and growinghyphae from already-germinated conidia were killed with heat at 90° C.for 1 h. The fungal material (killed conidia and hyphae) was pelleted bycentrifugation, resuspended in IntL-ELISA blocking buffer, and incubatedwith 10⁻⁷ M rIntL-1 (FLAG-tagged) in presence of 10 μg/mL of eitherMAb476 or an irrelevant, isotype control antibody mouse IgM for 2 h at30° C. Following washes with IntL-ELISA buffer, the material wasresuspended in IntL-ELISA blocking buffer and incubated with a 1:50diluted anti-FLAG tag antibody conjugated to the red fluorochromephycoerythrin (PE). Again, after washes, the stained material wasresuspended in IntL-ELISA blocking buffer, transferred to chamber slidesand observed under a fluorescent microscope.

The inventors performed experiments wherein Aspergillus ethanolprecipitate and whole cells (conidia and hyphae) were exposed to rIntL-1and then a sandwich assay using Rabbit polyclonal antibodies to hIntLand goat anti-rabbit antibodies with a fluorescent ligand were performedin buffer comprising 10 mM CaCl₂. As shown in FIG. 4, hIntL boundAspergillus ethanol precipitate with high affinity in the presence ofCa²⁺. Thus, hIntL could be the competitive agent which is interferingwith the inventors' antibodies to galF.

Example 2

Human IntL-1 is Present in Human Urine.

Studies were performed using urine collected from control patients (pool4) and patient who had Aspergillus infection (VU7). The samples were runin a reducing SDS polyacrylamide gel electrophoresis (PAGE), and theresolved proteins were transferred to a nylon membrane for performing aWestern Blot. Some samples used concentrated urine (denoted by “c”)using an Amicon concentrator or were pretreated by desalting columns(denoted by “ds”). As expected in a reducing SDS-PAGE, IntL (homotrimerin native state) was resolved to its monomeric form (MW range ˜25-40kDa). As shown in FIG. 5, hIntL was present in patient urine and couldalso be detected in healthy controls in the concentrated samples.

Example 3

Patient Urine Contains a Different Form of hIntL than Healthy Urine.

Following up from Example 2, Western blots using enzyme-labeled Rabbitantibody to hIntL-1 along with a chemiluminescent substrate corroboratethe fact that recombinant hIntL-1 is a 120 kDa homotrimer of about 40kDa subunits, as shown by bands generated due to partial and incompletereduction in the reducing SDS-PAGE. Monomers of native hIntL are knownto vary in 25-40 kDa range. Patient urine had significantly higherconcentrations (thicker bands) of small molecular weight subunits,whereas healthy urine had monomeric and dimeric subunits in the expectedsize range (FIG. 6).

Example 4

galF Specific Antibodies Recognize Certain Proteins in Urine.

The PEAKS search confirmed the presence of hIntL sequences in theanalyzed 476-reactive eluate, indicating the increased likelihood ofhIntL being the protein in the urine which competitively interferes withmAB476, and binds galF antigens.

Example 5

Testing of Various Methods to Inhibit hIntL Binding to galF Antigens inPatient Urine Samples.

As shown in FIG. 11, the inventors identified IntL-1 as binding to thesame Aspergillus derived galF-bearing antigens as mAB476 antibody. Sincedesalting the urine samples prior to contacting the sample with thegalF-reactive antibodies resulted in much greater binding and detection,it was surmised that removal of Ca²⁺ in the samples caused IntL-1 tolose its native ability to bind galF, making free galF much moreavailable to bind the galF-specific antibodies, such as mAB476. It wastherefore thought, that pre-treatment of urine samples with compositionsor compounds which either bound free mono and divalent cations, or whichserved as high-affinity hIntL-ligand, would allow the galF specificantibodies bind more of free galF in the samples.

As shown in FIG. 13, in an indirect ELISA assay where Aspergillusethanol precipitate containing galF is bound to the plate wells, agalF-binding antibody, such as mAB476, was added to the wells diluted inpooled healthy urine (pool 4), untreated or treated with compounds whichare known hIntL ligands or Ca²⁺ chelators; the samples with the knownligands or chelators had significantly greater antibody binding comparedto the untreated urine, and at the highest antibody concentration, thesignal was the same or better than the desalting (positive control fortreatment). Thus, the inventors have developed a successful methodologyto overcome hIntL competitive binding without use of extensivepretreatment processing of the urine samples.

As shown in FIG. 14, in a sandwich ELISA where a galF-binding antibody,such as mAB476, is bound to the plate wells as the capture antibody, lowconcentrations of Aspergillus ethanol precipitate, namely 0.1 and 1μg/mL, were diluted in urine, untreated or treated with compounds thatare known ligands of hIntL, or Ca²⁺ chelators, or a combination thereof,incubated with the same galF-binding antibody mAB476 conjugated toenzyme alkaline phosphatase (AP) as the detection reagent, and finallyplaced in the mAB476 coated wells. Following further incubation, thebound Ab-Ag-Ab complex was detected via color development using asubstrate of AP. It was observed that the samples with the known ligandsor chelators had significantly greater antibody binding signal at lowantigen concentrations compared to the untreated urine sample. Thisrecapitulates the success of the methodology developed by the inventorsto circumvent the hIntL competitive binding without extensive samplepre-treatment for detection of low antigen concentrations.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

The invention claimed is:
 1. A method for diagnosing a microbialinfection in a biological sample from a mammalian subject suspected ofhaving, having, or susceptible to having a microbial infection bydetecting the presence of at least one polysaccharide comprising agalactofuranose residue in a biological sample of the mammalian subject,the method comprising: (a) treating the biological sample to inhibithuman intelectin (hIntL) binding of galactofuranose residues present inthe sample, wherein the treatment of the biological sample comprisescontacting the biological sample with at least one ligand which bindsdirectly to intelectin, or substrates which bind divalent cations withhigh affinity; (b) contacting the treated sample of (a) with at leastone antibody specific for at least one polysaccharide comprising agalactofuranose residue in an effective amount to produce a detectableamount of antibody-polysaccharide complex, wherein the at least oneantibody specific for at least polysaccharide comprising agalactofuranose residue is selected from the group consisting ofmonoclonal antibody 205 (MAb 205) comprising a variable heavy (VH)domain of SEQ ID NO:1 and a variable light (VL) domain of SEQ ID NO:2;monoclonal antibody 24 (MAb 24) comprising a VH domain of SEQ ID NO:3and a VL domain of SEQ ID NO:4; monoclonal antibody 686 (MAb 686)comprising a VH domain of SEQ ID NO:5 and a VL domain of SEQ ID NO:6;monoclonal antibody 838 (MAb 838) comprising a VH domain of SEQ ID NO:7and a VL domain of SEQ ID NO:8; and monoclonal antibody 476 (MAb 476)comprising a VH domain of SEQ ID NO:9 and a VL domain of SEQ ID NO:10;and (c) detecting the presence of at least one antibody-polysaccharidecomplex, wherein the detection of the presence of at least oneantibody-polysaccharide complex is diagnostic of a microbial infectionin a mammalian subject.
 2. The method of claim 1, wherein in step (a)treating the sample comprises contacting the sample with a substrate. 3.The method of claim 2, wherein the substrate comprises a polyacrylamideresin.
 4. The method of claim 2, wherein the substrate comprises adesalting column.
 5. The method of claim 4, wherein the desalting columnhas a molecular weight cut off of less than 10 kDa.
 6. The method ofclaim 4, wherein the desalting column has a molecular weight cut off of7 kDa.
 7. A method for optimizing an assay for detecting the presence ofat least one polysaccharide comprising a galF residue in a biologicalsample of the mammalian subject, the method comprising: (a) treating thebiological sample comprising contacting the sample with a substratewhich binds salts with high affinity, thereby inhibiting humanintelectin (hIntL) binding of galF residues present in the sample; (b)contacting the treated sample of (a) with at least one antibody specificfor at least one polysaccharide comprising a galF residue in aneffective amount to produce a detectable amount ofantibody-polysaccharide complex, wherein the at least one antibodyspecific for at least polysaccharide comprising a galactofuranoseresidue is selected from the group consisting of monoclonal antibody 205(MAb 205) comprising a variable heavy (VH) domain of SEQ ID NO:1 and avariable light (VL) domain of SEQ ID NO:2; monoclonal antibody 24 (MAb24) comprising a VH domain of SEQ ID NO:3 and a VL domain of SEQ IDNO:4; monoclonal antibody 686 (MAb 686) comprising a VH domain of SEQ IDNO:5 and a VL domain of SEQ ID NO:6; monoclonal antibody 838 (MAb 838)comprising a VH domain of SEQ ID NO:7 and a VL domain of SEQ ID NO:8;and monoclonal antibody 476 (MAb 476) comprising a VH domain of SEQ IDNO:9 and a VL domain of SEQ ID NO:10; and (c) detecting the presence ofat least one antibody-polysaccharide complex.
 8. The method of claim 7,wherein the substrate comprises a polyacrylamide resin.
 9. The method ofclaim 7, wherein the substrate comprises a desalting column.
 10. Themethod of claim 9, wherein the desalting column has a molecular weightcut off of less than 10 kDa.
 11. The method of claim 9, wherein thedesalting column has a molecular weight cut off of 7 kDa.